Diphtheria toxin (DT) catalyzes the transfer of ATP-ribose from NAD to elongation factor 2 thus inhibiting protein synthesis and ultimately resulting in cell death. Sites within the DT A chain (DTA) were mutagenized and the resulting substitutions analyzed for their ability to inhibit enzyme activity. Two of these sites were identified based on CRM 228, a previously-isolated, enzymatically-inactive mutant. CRM 228 has tow mutations in DTA, G79D and E162K. These two sites were individually mutagenized and a variety of amino acids substituted. E162K was found to have no effect on catalytic activity while G79D reduced activity to background levels, demonstrating that the lack of enzymatic activity in CRM 228 was due to the G79D substitution. Analysis of the crystal structure of DTA reveals that E162 is located on the outside of the molecule while G79 is located along the enzymatic cleft.G52, also lies along the enzymatic cleft and is believed to be important in ADP- ribosyltransferase activity. This residue was substituted with a variety of amino acids and the enzymatic activity of the mutants analyzed. The majority of amino acid substitutions at this site significantly reduce catalytic activity.G52l and G79D mutations were selected based on their ability to reduce enzymatic activity and maintain native conformation. These sites together with H21G, a previously identified mutant with reduced enzymatic activity, were expressed individually and together in all possible combinations.